Study On The Autolysis Rules Of Trypsin And Its Autolysis-resistant Modification

Trypsin,a serine protease secreted in the pancreas,is widely used in scientific research,food processing,pharmaceutical and other industries.In the application,trypsin not only hydrolyze the substrate protein,but also hydrolyze itself.The autolysis greatly reduces its hydrolysis efficiency,and limits its application level.In addition,so far trypsin is produced mainly by extraction from animal pancreas,which have the disadvantages of limited material,high cost and low purity disadvantage.If it is produced by fermentation with microbiology,the production cost will be greatly reduced.This study tries to reveal the process and mechanism of autolysis of pig trypsin by mass spectrometry technology,improve its ability of autolysis-resistance through the molecular modification,realize its heterologous expression in microbiology by genetic engineering techniques.The methods and results are as follows:First,to express pro-trypsin by Pichia yeast,recombinant expression vectors pPIC9K-try was transformed into Pichia pastoris(P.pastoris)GS115,making methanol induction fermentation screened positive transformants found:broth supernatants SDS-PAGE in the presence of molecular weight controlled in line corresponding to strip compared,and activated by enterokinase detected after trypsin enzyme activity,indicating that the try of trypsinogen genes in Pichia was successfully expressed.Second,MALDI-TOF-MS and LC-Q-TOF technology were used to analyze the autolysis process of trypsin,and the most easily autolysis amino acid positions were identified as Lys-77,Arg-57 and Lys-97.Finally,the methylation cinditions of trypsin were investigated and optimized.The optimal methylation method were found as follow:the concentration of trypsin is 10 mg/ml,10 ?l 1M DMAB and 20 ? 1 M formaldehyde added for methylation for 3 h.The remaining activity of the methylated typsin after autolysis for 6 h reached 79%,which was about 18%more than the control.Findings of this study will help to strengthen the understanding of the autolysis of trypsin and improve its ability of autolysis-resistance.Furthermore it also sets the foundation for the production of trypsin with microbial fermentation in the future.